Abstract This grant application attempts to delineate molecular mechanisms that regulate the development and cell fate of the venous pole structures including the sinoatrial node (SAN) and the pulmonary vein (PV). We reported previously a key role for the homeobox gene, Shox2, in the development of the SAN. Mutation in Shox2 leads to hypoplasia and failed differentiation of the SAN as well as bradycardia in mice. While previous studies showed that Shox2 regulates SAN development through repression of Nkx2.5 expression in the SAN head domain, our recent study demonstrates that Shox2 and Nkx2.5 are co-expressed, together with Hcn4, in the developing venous pole, including the SA junction, a part of the SAN, and the PV myocardium, raising the possibility that the different parts of the SAN run distinct genetic program to regulate its development, and the SA junction shares similar genetic program with other venous structures. We found that reduction of Nkx2.5 dosage in Shox2 mutant background rescues SAN defects, and conversely, deletion of Shox2 in Nkx2.5 hypomorphic mice eliminates ectopic Hcn4 expression in the PV myocardium, suggesting the operation of a conserved Shox2-Nkx2.5 antagonistic mechanism in SA junction development and ectopic pacemaker formation in the PV. Physical interaction between Shox2 and Nkx2-5 and the extensive genome-wide co-occupancy of Shox2 and Nkx2-5 in developing hearts support a direct antagonistic mechanism. The fact that inactivation of Shox2 in the Nkx2-5+ SA junction and deletion of Nkx2-5 in the SAN cause sick sinus syndrome, indicating an essential role for both Shox2 and Nkx2-5 in SAN function. Most intriguingly, Shox2-null mice bearing Nkx2-5 deletion in the venous pole show normal pacemaker function, but exhibit a deformed SAN and high level of Hcn4 expression in the venous pole. Based on these observations, we hypothesize that Shox2-Nkx2.5 antagonism represents a common mechanism that regulates cell fate and pacemaker function in the venous pole, which is uncoupled from Shox2?s function in SAN morphogenesis. Three specific aims are proposed to test this hypothesis: 1) to establish the heterogeneous development and functional model of the SAN; 2) to dissect genetic modules that distinctly regulate SAN morphogenesis uncoupled from venous pole pacemaker program; 3) to decipher the underlying mechanism of Shox2-Nkx2.5 antagonistic machinery in venous pole development. The proposed studies will systematically address the functional mechanism of Shox2 and Nkx2.5 in pacemaker development including ectopic pacemaker in the venous pole and dissect the physiological function of the two distinct SAN domains through a combination of genetics, morphometric, molecular biology, genomics, and electrophysiological approaches. Since SHOX2 was recently identified as a susceptibility gene associated with atrial fibrillation in patients, the results obtained will provide novel insights for a better understanding of the molecular regulatory mechanisms of pacemaker development and the formation of ectopic triggers, and shed light on the etiology and design of gene- and cell- based therapeutic approaches to congenital and acquired cardiac conduction system abnormalities.